Oxysterols are molecules that cells synthesize from cholesterol, which affect many biological processes such as metabolism, cell-cell signaling and cellular migration. Surprisingly, in spite of their potent activities, we understand only poorly th roles that oxysterols play, and how they act to accomplish various effects on cells. Oxysterols have been implicated in Hedgehog signaling, a cell-cell signaling pathway essential for embryonic development, for adult stem cell maintenance, and deeply involved in many human cancers. It is known that oxysterols bind and activate a membrane protein, Smoothened, which is critical for triggering Hedgehog signal transduction. However, many critical aspects of the role of oxysterols in Hedgehog signaling have remained obscure: we do not know if oxysterols are required for normal Hedgehog signaling, how they are regulated, and whether blocking oxysterols might be a good way to inhibit Hedgehog signaling in cancer. Additionally, there is a need for new and powerful chemical approaches to visualize and assay oxysterols in vivo; such methods would greatly aid our understanding of their role in Hedgehog signaling and beyond. We developed novel chemicals, called azasterols, which block binding of oxysterols to Smoothened; as a result, Hedgehog signaling is inhibited, which shows that antagonizing oxysterols is an effective strategy to inhibit the Hedgehog pathway. Using azasterol and oxysterol chemical probes, we pinpointed where oxysterols bind to Smoothened, which allowed us to build Smoothened mutants that no longer respond to oxysterols. Interestingly, we discovered that these Smoothened mutants cannot signal properly; this showed that oxysterols are required for normal Hedgehog signaling. Finally, we have synthesized and characterized novel chemical probes that allow us to visualize and assay oxysterols in cells, with better sensitivity and specificity than before. We plan to use these probes to better understand the role of oxysterols in Hedgehog signaling. We propose to use a combination of chemical biology, biochemistry and cell biology, to accomplish the following aims: A) To elucidate how Hedgehog signaling regulates oxysterols, with the aid of our novel oxysterol probes B) To determine precisely which oxysterols are involved in Hedgehog signaling C) To discover how oxysterols involved in Hedgehog signaling are synthesized in cells These studies are important for the following reasons: 1) They will advance our understanding of Hedgehog signaling, by elucidating the critical role of oxysterols; 2) They will identify novel targets for blocking Hedgehog signaling in cancer, based on oxysterol inhibition; and 3) Our novel chemical probes will be broadly applicable to study oxysterol mechanisms in health and in disease, will provide diagnostic tools for sterol disturbances and will help identify small molecule inhibitors of sterol function.